During development, the vertebrate central nervous system is formed by partitioning proliferating primordial cell populations into compartments and gradually refining these populations into distinct functional sub- regions. In the presumptive midbrain and hindbrain, these compartments are lineage restricted by a sharp boundary interface ensuring that cells from these regions do not mix and that they receive region specific signals to properly develop into the tectum and cerebellum, respectively. These region specific signals originate from an organizer, the isthmic organizer, positioned at the midbrain-hindbrain boundary (MHB). Though many of the key regulatory signals of the isthmic or MHB organizer are known, our current understanding of how these regions are initiated, formed, and maintained remains poor. Furthermore, developmental defects of these regions and brain stem have been linked to conditions such as intellectual disabilities, autism, and Chiari malformation. A detailed study of human brain formation in utero to better understand its development and developmental disorders is problematic ethically, but the MHB is one of three evolutionarily conserved organizers of the vertebrate brain. Thus, understanding how the MHB forms in model species such as zebra fish will likely provide valuable insights into human brain development, particularly the tectum and cerebellum. How- ever, the technological capability and methodology to characterize the dynamics and interactions of developmental lineages in the early zebra fish embryo has yet to be demonstrated. This project will develop the technologies and reagents that address fundamental challenges to characterizing the dynamics and interactions of distinct developmental lineages in the zebra fish midbrain and hindbrain and that may be extended to other functional regions and developmental stages and, potentially, other model species. The MHB forms in the zebra fish at an early developmental stage of rapid cell movement through 3D space. At this early developmental stage, cells and their identities are morphologically indistinct and may only be differentiated by gene expression. Thus, a fundamental challenge in characterizing dynamics and interactions of presumptive midbrain and hindbrain cells in MHB formation has been the capability to acquire volumetric images in developing embryos fast enough and with high enough spatial resolution to resolve single cells and to identify the imaged cells by the expression of key regulatory genes. Aim 1 of this project generates transgenic zebra fish with fluorescent reporters that fiducially mark future midbrain and hindbrain cells. Aim 2 of this project develops novel light sheet microscopy based on ultra-short optical pulses. Time-lapse, multimolecular light sheet microscopy will be used to test two main hypotheses: the initially overlapping boundary of mid- brain and hindbrain cells is sharpened through cell sorting; and lineage restriction properties of the MHB are established coincidentally with sharpening of the boundary interface.